Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells

Definitions

• the present invention relates generally to phenotyping and immunophenotyping of cells and more particularly to single parameter and multiparameter phenotyping and immunophenotyping of cells.
• Immunophenotyping of cells and tumors, particularly hematopoietic tumors, is often of critical importance for clinical evaluation of cancer patients.
• currently available methodologies particularly flow cytometry, are expensive and require a high degree of suspicion at the time of biopsy. All too often, even before the diagnosis of cancer is made, precious tissue must be set aside for possible immunophenotyping. If tissue is not set aside and there is cancer present, the correct subtyping of the tumor (and proper assignment to treatment protocols) cannot be done after the fact. Methods that do not require forethought, such as immunostaining of paraffin blocks, are far less sensitive and do not work well in laboratories that do not perform these stains frequently.
• Flow cytometry is the currently accepted "gold standard" for immunophenotyping of hematopoietic cell types.
• the expense of establishing and maintaining these laboratories is perhaps the most severe problem.
• Generally large hospitals, academic centers, or commercial reference laboratories are the only institutions capable of establishing flow cytometry laboratories. These laboratories often charge a premium for their services, and transportation of specimens to laboratories is not a trivial problem.
• Since flow cytometry requires live cells, specimens must be handled under sterile conditions. In laboratories where the technology is unavailable, a fresh specimen has to be prepared and shipped to a flow cytometry laboratory under sterile conditions for evaluation. Uncontrollable factors such as temperature variations, rough handling, bacterial contamination, or shipping delays may render samples unsuitable for analysis.
• flow cytometry requires technologists who have specialized training and their time must often be dedicated solely to the technology itself, further increasing the expense of the procedure. Relatively large volumes of cells must be analyzed in order to obtain statistically meaningful results during analysis.
• red cells must be removed from the sample prior to analysis. This is because the number of red cells in blood and bone marrow samples is far greater than other cells types, and shear numbers alone would overwhelm the sensitive detectors of the machines.
• the sample preparation method therefore requires Ficoll-Hypaque separation, followed by multiple washes, followed by a lysis step to lyse remaining red cells.
• Antibodies are proteins produced by the body's immune system that have the property that they bind to a singe specific molecule (referred to as an antigen). Antigen-antibody complexes are formed when an antibody binds its respective antigen. Normally, these complexes are then cleared by the immune system to rid the body of an infection.
• the immune system has a virtually limitless capacity to produce unique antibodies, which can be tailored to identify particular substances, even when present in very small quantities.
• Antibodies are now commercially produced to literally hundreds of different antigens.
• marker molecules such as fluorescent molecules, dyes, or other substances that make identifying the presence of an antigen-antibody complex a relatively simple matter.
• This well- known biochemical reaction has been used to develop a methodology called flow cytometry. In flow cytometry, intact cells are treated with antibodies that bind specific markers on the cell surface. The antibodies are, in turn, labeled with a fluorescent molecule and the cell suspension then flows past a light beam with a light detector which counts the number of fluorescent cells versus the other cells present.
• Both single parameter and multiparameter analysis can be performed. If data is analyzed as histogram plots of fluorescence of a single marker versus cell number, then one parameter analysis is being performed. Analyzing two such histograms of a single gated cell population could then be referred to as simultaneous single parameter analysis. An example of simultaneous single parameter analysis would involve the use of such plots to identify cell surface expression of both the B-cell marker CD20 and the light chain kappa. Analysis of the binding of each set of antibodies is independent of the other. In multiparameter analysis, the binding of the two antibodies are linked and are not independent. Analytical methods require the binding of both antibodies simultaneously brought together in a single histogram such as fluorescence 1 versus fluorescence 2.
• Characterization of the target cell population is best performed by analysis of this fluorescence 1 vs. fluorescence 2 plot and analyzing the binding characteristics of each of these antibodies together. This decreases the possibility of an error that would incorrectly analyze two overlapping cell populations as a single cell population.
• characterization of solid tumors and non-hematopoietic tumors is quite limited by flow cytometry. Often there are not well developed protocols for developing cell suspensions. In addition, tumor cells may be delicate and may not survive processing. In addition, many markers used for solid tumors such as vimentin or smooth muscle actin are intracytoplasmic antigens and may be difficult to assay by flow cytometry.
• An object of the invention is to provide a cheaper, more accessible method for single parameter and multiparameter analysis of cell populations.
• This analysis is not limited to just cell surface markers but also optionally includes identifying active receptor sites on cell surfaces, loss of cell surface proteins, intracellular proteins, and intracellular nucleic acid sequences.
• One of the features of this invention is that the target cell population is being analyzed by preserving mo ⁇ hologic characteristics of the cells for analysis.
• a method of characterizing cells comprising the steps of a) providing a suspension of cells in a liquid medium, said cells including first cells, b) adding to said suspension a group of substantially identical first beads, each of said first beads being coated with a binding substance or being magnetic such that each first bead is adapted to bind to a first cell, c) incubating said first beads in said suspension for a period of time effective to permit said first beads to bind to said first cells to form first bead-first cell complexes, each first bead-first cell complex comprising a first bead and a first cell, d) separating said first bead-first cell complexes from said suspension by filtration, and e) examining said separated first bead-first cell complexes and characterizing said first cells.
• a kit comprising at least one group of substantially identical first beads, each of said first beads being coated with a binding substance or being magnetic such that each first bead is adapted to bind to a first cell, said kit further comprising a set of instructions effective to instruct a technician in how to use said first beads to perform single parameter or multiparameter analysis on a suspension containing first cells.
• An apparatus for performing single parameter or multiparameter analysis on a suspension of cells comprises a sample loader, a plurality of reaction chambers, and a filtration chamber.
• Fig. 1 is a schematic illustration showing a cell bound to an antibody which is bound to or coated on a bead.
• Fig. 2 is a schematic illustration showing a number of cells bound to a bead.
• Fig. 3 is a schematic illustration showing a number of cells bound to a bead in the center, and five smaller beads bound to five of the cells.
• Fig. 4 is a schematic illustration of an automated device for performing phenotypic or immunophenotypic analysis in accordance with the present invention.
• Fig 5. is a schematic illustration of a single slide for use with the automated device of Fig. 4.
• a preferred range such as 5-25 this means preferably at least 5 and, separately and independently, preferably not more than 25.
• the cells herein are preferably human cells If a first group of cells does not include members of a second group of cells, and the second group of cells does not include members of the first group of cells, the two groups do not overlap. "Visually distinguishable” includes visually distinguishable via light microscopy. Quantitate includes to estimate or enumerate or count the number of. Phenotyping includes immunophenotyping and genotypmg. The invention uses beads.
• beads means small particles or support surfaces, preferably microspheres, more preferably plastic microspheres, more preferably polystyrene microspheres (also referred to as latex beads or spheres or microspheres), which have preferably been coated with a binding substance or which are magnetic.
• first beads includes beads which have been coated with a binding substance or which are magnetic.
• the bead may be any solid support surface or particle that can be suspended in an approp ⁇ ate solution.
• Preferred beads are available as polystyrene microshperes from Bangs Laboratories, Fisher, IN.
• the bead sizes are preferably greater than 5 microns diameter, preferably 5.5-10.3 microns, less preferably at least 5.5 or 10 or 12 microns and preferably not more than 12, 15, 20 or 30 microns diameter, far less preferably less than 5 microns, such as at least 1, 2 or 3 microns diameter.
• 5.5 and 10.3 micron beads are preferred.
• the beads can also be colored, such as red or blue, less preferably green, pu ⁇ le, orange, brown, yellow, or any other color.
• the beads are preferably coated with a binding substance, such as antibodies or immunoreactive proteins, or any molecule that can bind to, or interact with a cell surface in such a way as to bring the cell and the bead into contact or adherence with each other or to bind with each other; alternatively, the bead can contact or bind with the cell surface through electrostatic charge interactions or magnetic interaction; all of these concepts being covered by the terms "bindmg to" or "bind to"
• a bead binds to a cell, it forms a bead-cell complex
• the cell- bead interaction forms a large enough complex to inhibit the passage through a filter containing pores of approp ⁇ ate dimensions.
• the filters are preferably sized and selected such that unbound cells and beads will pass through the pores but bound cell-beads do not
• These complexes are then transferred to a glass slide and stained with a va ⁇ ety of stains so as to render the complexes visible by routine microscopy.
• the complexes and cells are examined and the cells are characte ⁇ zed
• binding substances or reactive substances that may be used to coat the bead surface mclude, but are not limited to: antibodies to specific cell surface proteins, small molecules that b d receptors or other cell surface molecules such as IL-2 or GM-CSF, avidin, biotin, or beads may remain uncoated in suspension that can interact by other means with cells
• the antibodies that can be used are those known in the art.
• Beads or microspheres can be made from a variety of substances including gold, ferritin, polyacrylamide, or polystyrene. The latter is among the preferred substances as beads can be made precisely to any size specification and can be uniformly conjugated to both molecular linker arms and reactive binding substances.
• Polystyrene microspheres also known as "latex microspheres" may be prepared by methods known in the art which are inco ⁇ orated by reference herein.
• Binding substances that can be used include monoclonal antibodies, polyclonal antibodies, antibody "cocktail” mixtures, antibody fragments (such as Fc portions or Fab or Fab' fragments in either monovalent or divalent forms), small molecules that bind specific cell surface receptors, covalent and non-covalent linkers, and indirect adherence such as utilizing electrostatic or magnetic or paramagnetic attraction.
• the prior art includes U.S. Pats. 5,554,505; 5,348,859; 5,340,719; 5,231 ,005; 5,260, 192; 5,338,689; 5,256,532; and 5,501,949, the entire contents of which are inco ⁇ orated herein by reference. These patents include discussions of using certain microspheres or beads for identification of cells.
• a second feature of a preferred embodiment of the present invention is that it concentrates cells by using an appropriate filter without added manipulation of the cell suspension by cell lysis or added incubation steps of submicroscopic paramagnetic microspheres.
• the filters to be used in the invention can be any of those known in the art, such as gynecological filters from Cytec Co ⁇ . , Boxborough, MA.
• the filters preferably have a pore size larger than the beads being used so that all or most or substantial amounts of unbound beads and unbound cells pass through, but the pore size is preferably small enough so that all or most or substantial amounts of beads bound to cells are trapped on the filter, such as the filter pore size being about 1 , 2, 3, 4, 5, 6, 8, 10 or 12 microns larger than the bead size.
• Preferred filter pore sizes include 10-15, less preferably 7-20, 7-30 or 7-40 micron pore sizes.
• the filter pore sizes can be at least 15 or 20 microns.
• the filter is preferably mounted on a solid support, such as at the end of a tube through which the suspension can drain.
• a cell suspension is preferably prepared from a peripheral blood sample, a bone marrow aspirate, a fine needle aspirate, a lymph node biopsy, or a body site specimen.
• single parameter, simultaneous single parameter, and true multiparameter analysis is possible which compares to the level of sophistication of analysis possible by flow cytometry.
• Beads that can be easily distinguished from each other optically either by size, color, or both can be added to a cell suspension either simultaneously or sequentially. Positive binding by the target cell population results in a bead-cell complex that has a significantly larger physical size than either unbound cells or beads.
• MDS myelodysplastic disorders
• a bead 2 such as a polystyrene microsphere, which has coated thereon and bound thereto a binding substance 4 such as an antibody.
• a cell 6 such as a target cell, which has a cell surface marker 8. The binding substance 4 or antibody binds to the cell surface marker 8 on the target cell 6.
• FIG. 2 illustrates how this kind of reaction may appear on a glass slide; a group of cells or target cells 12 have bound to a bead 10.
• This shows single parameter binding of cells to beads.
• the ratio of beads to cells should be adjusted properly for effective results.
• the actual number of cells binding the bead is variable, ranging from a single cell to numerous cells crowding the bead's surface.
• Fig. 3 there is shown a large bead 14 coated with a binding substance which has bound to eight cells 16, 18, 20, 22. Small beads or different colored beads 24 coated with a different binding substance have bound to the cells 22 but not to the cells 16, 18, 20. This provides positive identification of target cells 22.
• Cells 22 is a subset of cells 16, 18, 20, 22.
• a variable number of beads 24 can bind to each cell 22.
• each cell bound to bead 14 will be bound to one or more beads 24, or each cell bound to bead 14 may be unbound to small beads.
• different kinds of cells may bind to the large bead 14 that can in some cases be distinguished mo ⁇ hologically.
• the large bead 14 is added first to the cell suspension so that a plurality of cells can bind to its surface.
• the small beads 24 are added to bind to the periphery of the complex.
• small beads 24 can be added first or small beads 24 and large beads 14 can be added simultaneously. The order of addition is dependent in large part upon the relative concentrations and surface areas of the beads and the cells.
• the suspension can be filtered after the first complex is formed, to trap the first complex and resuspend it before the second beads are added.
• a group of complexes can be filtered and resuspended before a subsequent set of beads is added; this can lead to more certain and distinct results by removing materials which would provide interference.
• the beads may be distinguishable in size or color or both. Further levels of multiparameter analysis can also be carried out, such as by adding to Fig.
• Colorless 10 micron beads are used to bind B cells by using anti-CD 19 coated beads.
• 5-micron colorless beads are coated with anti-kappa while dark blue 5-micron beads are coated with anti-lambda.
• a blast cell population can be analyzed using anti-CD34 coated 10 micron beads and anti-CD19 coated colorless 5 micron beads.
• Colored 5-micron beads coated with anti-CD 13 are simultaneously added for rapid characterization of most blast cell populations.
• Preferred methods 1) Substantially identical beads are purchased commercially precoated with strepavidin (Bangs Laboratories, Fishers, IN). A small quantity is suspended in any buffered salt solution such as phosphate buffered saline or commercially available antibody diluent.
• the beads are incubated with biotinylated goat anti-mouse antibodies for 30 minutes (however, any biotinylated anti-allogeneic antibody may be used).
• the suspension is centrifuged and the supernatant drawn off. The incubation is repeated two times to ensure coating of as much of the available surface area of the beads as possible.
• the beads are then washed three times using the same buffer.
• the suspension is then incubated with specific mouse anti-human antibodies for 1 hour (or any non-biotinylated anti-allogeneic antibody specific for the target cell population may be used).
• the suspension is again washed three times and diluted to the desired concentration.
• the resulting suspension can be refrigerated at 4 degrees Centigrade until use.
• biotinylated primary antibodies may be used without the use of secondary antibodies.
• the beads produced by this technique are substantially identical.
• Beads are precoated with anti-Fc receptor antibodies (Bangs Laboratories, Fishers, IN) such as goat anti-mouse IgG Fc receptor antibodies. These beads can then be suspended in a solution of antibodies which would spontaneously bind to the anti-Fc receptor sites on the beads.
• anti-Fc receptor antibodies Beads Laboratories, Fishers, IN
• anti-Fc receptor antibodies Bos Laboratories, Fishers, IN
• Binding substances such as any protein, peptide, or nucleotide sequence may be bound by other chemical or specific binding methods.
• polystyrene microspheres are "naturally" left coated with sulfate surface groups after manufacture.
• These ligands can be used to link proteins and peptides directly to the surface of the beads.
• functional surface groups that can be coated on the surface includes, but is not limited to, aldehyde, aliphatic amine, amide, aromatic amine, carboxylic acid, chloromethyl, epoxy, hydrazide, hydroxyl, sulfonate, and tosy (toluene sulfonyl) reactive ligands. These can then be used in turn to link peptides, proteins, oligonucleotides, and other biochemical ligands to the surface.
• ligands or binding substances would in turn be used to bind specific sites on cell surfaces which would link the cell to the surface of the bead.
• a small molecule such as the hormone IL-2 could be used by one of the above methods to coat beads with the intention of binding IL-2 receptor sites (CD25) on cell surfaces.
• This could be used to bind cells such as T-cells, monocytes, and neoplastic cells such as hairy cell leukemia.
• Submicroscopic paramagnetic microspheres are bound to any reactive biomarker of interest. The binding that is used could be any of the above methods.
• Cells are then permeabilized and fixed using a variety of detergents and weak fixative solutions such as 1 % paraformaldehyde.
• a number of commercially available permeabilizing kits are available for this pu ⁇ ose such as IntraStain (Dako Co ⁇ . , Ca ⁇ interia, CA).
• the reactive biomarker such as antimyeloperoxidase antibodies, anti-terminal deoxytidyl transferase antibodies, or specific RNA or DNA probes, is then incubated with the cell suspension.
• the biomarkers and paramagnetic particles get inside the cell and, for example, the probe binds to the intracellular target.
• the cells are then washed and resuspended in a suitable buffer such as PBS or RPMI.
• a suitable buffer such as PBS or RPMI.
• the suspension is then incubated with magnetic beads or microspheres of a size or color easily visualized, such as 1 to 20 or 3-15 or 5-10 or 10-20 microns.
• the magnetic beads bind to the cell surface, but cannot cross the membrane, to create a cell-bead complex that is easily trapped such as via filtration.
• abnormal blasts in a bone marrow suspension can be permeabilized and incubated with anti-myeloperoxidase antibodies bound to submicroscopic paramagnetic microspheres.
• the suspension is then washed three times in buffered salt solution and resuspended and incubated with large magnetic beads of a preferred size of 5-15 micron diameter to create cells bound to large beads.
• specific DNA sequences probes
• Cells from a patient with chronic myelogenous leukemia are permeabilized and incubated with probes binding to the specific bcr-abl translocation that is diagnostic for the disease.
• the suspension is then washed and incubated with large magnetic beads of a preferred size of 5-15 micron diameter to create cells bound to large beads.
• the cell-bead complexes (cells bound to beads) provided or obtained as described above are then passed through a solid support filter having a porosity of sufficient size to allow unbound cells and beads to pass through.
• the suspension is passed through the filter using a variety of acceptable methods which includes gravity, suction (applied vacuum), positive pressure on the fluid side, or wicking the fluid through the filter using a porous absorbable material such as gauze pads.
• Various devices that can be used include pistons, syringes, or suction methods to create a negative pressure to pass fluid through the filter.
• a single solid filter with a pore size of 10-15 microns is used.
• Cell-bead complexes remain trapped on the filter and the layer is then transferred to a glass slide by direct contact with the slide and applying gentle pressure.
• the resulting slide preparation can be stained using a variety of commercially available stains such as hematoxylin and eosin, Papanicolau stain, or any Romanowsky stain.
• the cells remain suspended in a compatible buffer such as PBS, RPMI, or commercially available antibody diluent and the resulting slide is stained with Wright-Giemsa stain.
• cells may be suspended in ethanol or a commercially available fixative such as Cytolyte (Cytyc Co ⁇ ., Boxborough, MA).
• the resulting slide is then stained with Papanicolau or hematoxylin and eosin stains.
• the complexes are examined and the cells are characterized under routine light microscopy.
• the invention can be used to perform single parameter analysis correlated with mo ⁇ hology, simultaneous single parameter analysis, or multiparameter analysis.
• single parameter analysis (depicted in Figs. 1 and 2) a single bead type is added to a suspension of cells in a liquid medium so that after filtration the slide is provided with an enriched single cell population. This is useful as a simple screen to determine if a cell population has a particular characteristic such as distinguishing monocytes from monocytoid B lymphocytes as cited in Example 1 below.
• a B cell population can be assayed for expression of kappa or lambda by using two separate slides or slide wells each of which contain a single bead type (anti-kappa or anti- lambda).
• Another variant of this analysis is to add simultaneously to the cell suspension two different bead types, one anti-kappa and a second anti-lambda. This is an example of simultaneous single parameter analysis since binding of each bead type is independent of the other but the results are analyzed together.
• An analogous situation occurs in flow cytometry analysis when fluorescence is displayed vs. cell number to obtain a single histogram.
• a monoclonal population can only be detected by simultaneous analysis of both histograms and looking for single peaks of fluorescence.
• multiparameter analysis can be performed by linking detection of two different characteristics so that analysis is performed together. In this case, binding of one set of beads occurs, followed by a second and optionally more sets of beads (see Figure 3). Analysis looks for simultaneous binding of more than one set of beads to the target cell population (as depicted in Example 2 below).
• the invention can be used to detect abnormal loss of binding when strong binding would be expected. For example, normal myeloid cells such as mature granulocytes and monocytes in the peripheral blood would be expected to strongly express the surface markers CD13, CD33, CDl lb, and CD 16.
• a complementary detection method is that p ⁇ or to transfer of the cells to a glass slide, the filter is gently rinsed and scanned using a light beam of either a white light beam or a specific wavelength to correspond to the excitation wavelength of fluorescent beads The number of events is counted electronically and the cells are then transferred to a glass slide and stained. The average number of cells per microsphere is then obtained manually and an estimate of the total number of target cells in the sample can be estimated (assuming that a known volume of sample is used).
• Preferred applications 1) Single parameter analysis of tumors and other specific cell populations. A suspected tumor with a known immunophenotype can be analyzed to confirm the presence of a single marker as outlined in Examples 1 and 3 below.
• Example 1 knowing that the abnormal cell population is of B cell o ⁇ gm is sufficient information to proceed with further studies, since this suggests (but does not prove) malignancy.
• Example 3 knowing that the lymphoid population is of T cell o ⁇ gm suggests that the patient has a reactive infiltrate rather than a malignant infiltrate. If this assay had been clinically available in both of these unusual cases, the results of the simple study in Example 1 would justify further expense of additional evaluation. The results of Example 3 justify not performing flow cytometry and proceeding to treatment for meningitis.
• MN/CA9 screening for cervical cancer identifying specific tumor types in malignant infiltrates such as melanoma (using markers such as HMB-45), or identifying micrometastic disease in lymph nodes and bone marrows.
• single parameter analysis can be used in genetic phenotypic and genotypic analysis.
• a pe ⁇ pheral blood sample can be permeabilized and treated with a specific probe to the bcr-abl translocation.
• the probe can be labeled with paramagnetic submicroscopic microspheres.
• the cells can then be treated with large, magnetic beads to identify the presence of the translocation that would be diagnostic of chronic myelogenous leukemia.
• a similar method can be used to identify the presence of intracellular proteins or RNA sequences using approp ⁇ ate antibodies or nucleotide sequences, for example, the expression of the intracellular protein terminal deoxy ⁇ bonucleotidyl transferase (TdT) using an antibody also labeled with paramagnetic microspheres and detecting the reaction using large surface magnetic beads.
• TdT intracellular protein terminal deoxy ⁇ bonucleotidyl transferase
• CD64 expression has been proposed as a rapid diagnostic test for clinically significant acute inflammatory reaction (Lab. Hematol. 1995; 1 :3-12). For reasons desc ⁇ bed above, flow cytometry is too expensive and difficult to use as a screening procedure for common conditions.
• the invented method allows rapid, inexpensive single parameter analysis for CD64 expression in pe ⁇ pheral granulocytes.
• Simultaneous single parameter analysis is where there is simultaneous analysis of markers that are independent of each other. Most commonly, this is used in a B cell lymphoid population to determine expression of either kappa or lambda light chain rest ⁇ ction by expressed surface immunoglobulins. This can either be done by using similar beads as used in two separate glass slides analyzed simultaneously or by using a single slide using two sets of beads which can be easily distinguished based on size, color, or both. This is extremely useful as an inexpensive, rapid screen for B cell monoclonality.
• Example 2 A simple, but common, example of this kind of analysis is depicted in Example 2 below.
• the positive binding reaction by the anti- CD20 coated beads which isolates the B cells is linked to kappa or lambda light chain expression.
• Multiparameter analysis enhances analysis since correctly identifying certain cell populations requires logical association of multiple subsets of markers.
• a case of acute leukemia serves as a useful example of this kind of analysis
• Mo ⁇ hologic examination is one of the best methods for identifying the abnormal blast cells, but it does not characte ⁇ ze the kind of blasts present.
• Combining mo ⁇ hologic analysis with the present invention would yield the following typical kind of analysis.
• Ant ⁇ -CD34 coated beads are combined with anti-HLA-DR coated beads to confirm expression of both of these markers in the malignant cell population.
• the cells can then be analyzed with anti- CD13 and ant ⁇ -CD33 coated beads in conjunction with ant ⁇ -CD19 and ant ⁇ -CD2 coated beads to determine if the cells are myeloid or lymphoid in o ⁇ gin. If they bind to CD13, CD33, or both, this confirms the myeloid de ⁇ vation of the cells.
• the cells can also be analyzed with ant ⁇ -CD15, ant ⁇ -CD14, ant ⁇ -CD56, ant ⁇ -CD7, and ant ⁇ -CD4 to determine subtype (myeloid, monocytic, or both) and to yield prognostic information.
• ARB subtype M3 acute promyelocytic leukemia
• FAB subtype M3 acute promyelocytic leukemia
• flow cytometry is fraught with errors and the tumor can be missed since it is composed of matu ⁇ ng myeloid cells.
• mo ⁇ hologic analysis would confirm the presence of excess numbers of promyelocytic cells.
• the promyelocytes would usually be HLA-DR negative and could also be analyzed for the translocation of chromosomes 15 and 17 (t(15; 17)) which is diagnostic of the disease. This kind of analysis is particularly useful in the ⁇ ucrogranular va ⁇ ant of the disease in which the cells may resemble monoblasts.
• Monocytic leukemias can also be analyzed for additional monocytic markers such as CD36. Similar kinds of analyses can be performed for other hematologic malignancies, other tumor types, and other specific cell populations.
• the method can be used in reverse to offer a diagnostic test for myelodysplasia. Normal myeloid cells strongly bind the myeloid markers CDl lb, CD13, CD16, and CD33. Among the changes seen in myelodysplasia, is decreased expression of these markers by flow cytometry. However, degenerating cells, as occurs in excessive sample age, temperature extremes, or other forms of specimen mishandling also causes decreased expression of these markers.
• Her2/neu cytoplasmic membrane expression is estimated by the observer visually on a scale expressed as 0+ positive (no expression) to 4 + positive (strongest possible expression). There are no objective quantitative methods to estimate the level of Her2/neu overexpression.
• Her2/neu expression can be more objectively estimated by using fluorescent m-situ hyb ⁇ dization (FISH) which labels each gene copy with a fluorescent dot. The number of gene copies in each cell can be estimated by merely counting the dots within the nucleus of each cell.
• FISH fluorescent m-situ hyb ⁇ dization
• Her2/neu expression can be performed by flow cytometry, however, like FISH there is no method for evaluating whether the analyzed cell is a malignant cell or a benign one.
• epithelial cells in a cell suspension can be distinguished from stromal cells by using large (10 micron) beads coated with anti-cytokeratin antibodies. Only epithelial cells would bind to this bead. Small 5 micron beads coated with an appropriate anti-Her2/neu antibody is then added to the mixture and the suspension filtered.
• Her2/neu expression can be analyzed objectively by several methods.
• the filter itself can be analyzed to determine the quantity of 5 micron beads present on the filter by using methods such as fluorescence (if the 5 micron beads are fluorescent), electrostatic assessment, or other of a variety of known counting methods.
• the suspension is transferred to a glass slide after filtration and the slide stained.
• Benign cells can be distinguished from malignant ones by mo ⁇ hologic assessment and the average number of beads binding to malignant cells can be estimated. This can either be performed manually by the observer or in a semi -automated manner using an electronic visual analysis to count the number of beads bound to each cell identified by the observer as malignant. 4) Signal amplification of weakly expressed antigens.
• One of the major advantages of flow cytometry is its ability to detect weakly expressed antigens on the surface of cells. Many antigens fall under this category and cannot be easily detected using alternative means such as routine immunostains using standard colorimetric detection methods such as diaminobenzadine (DAB).
• DAB diaminobenzadine
• This problem in immunostains has been partially overcome using signal amplification methods such as tyramide signal amplification which is commercially available such as the Catalyzed Signal Amplification kit (Dako Co ⁇ ., Ca ⁇ interia, CA).
• the primary antibody is conjugated to peroxidase enzyme (usually horseradish peroxidase or HRP) and oxygen free radicals are generated.
• tyramide In the presence of tyramide, the tyramide molecules themselves become free radicals and are short lived, highly reactive species. They readily conjugate to nearby molecules and are fixed in the immediate area of the primary antibody.
• the signal amplification derives from the ease in which tyramide is conjugate either to a fluorescent molecule or peroxidase. This added peroxidase is used to generate additional DAB signal and thus the signal is amplified.
• This signal amplification technique can also be applied to the invented method described herein.
• primary antibodies are conjugated to HRP to generate biotinylated tryamide free radicals as per the manufacturer's directions. Avidinated beads then readily and spontaneously bind to the cell surface at the appropriate sites.
• An alternative method uses submicroscopic beads that are invisible by routine light microscopy which are coated with the antibody of interest that also have a peroxide free radical generator such as HRP bound either to the antibody or to the surface of the bead.
• Biotinylated tyramide free radicals are generated as per the manufacturer's directions and then the cells are washed (or filtered) and treated with avidinated large beads that are easily visible by light microscopy (typically beads in the 5-20 micron size range).
• This method of signal amplification greatly enhances otherwise weak binding of beads when only rare antigens are present on the cell surface.
• Single amplification can also be achieved using (1) the dual-labelled Envision polymer system available from Dako Co ⁇ ., Ca ⁇ interia, CA.
• An alternative method of multiparameter analysis can be performed by first using a single set of beads to isolate the target cell population.
• the second parameter can then be detected by using routine or conventional immunohistochemical techniques such as immunflouresence, colorometric methods such as peroxide reduced DAB or alkaline phosphatase methods, or immunogold/silver enhancement.
• This second antibody detection system can be applied either in the cell suspension or after the slide is made but before it is stained. The choice of method and detection method would be dependent on the desired stain in the final product and the particular antibody to be used.
• Example 1 A 30 year old man presented with pancytopenia and splenomegaly. Examination of the peripheral smear confirmed the pancytopenia. In addition, scattered cells were present that showed bland cytological characteristics, with a monocytoid appearance. The nuclei of these cells were round to oval, with a single intermediate nucleolus. There was abundant blue-gray cytoplasm that showed numerous cytoplasmic projections. A bone marrow examination revealed a hypocellular aspirate with similar cells present.
• Example 2 A 68 year old man with a known history of chronic lymphocytic leukemia (CLL) presented for routine follow up examination.
• Clinical examination revealed that the patient had a peripheral white cell count of 435,500 cells/ml (normal range 4,300-11 ,000 cells/ml) which included 87 % lymphocytes.
• Mo ⁇ hologic examination of the peripheral blood smear revealed predominantly an abnormal population of small lymphocytes with a small but significant population of large transformed cells.
• a suspension of cells in a liquid medium was provided. This sample was analyzed using anti-CD20 coated 10-micron beads, anti-kappa coated colorless 5-micron beads and anti-lambda coated colorless 5-micron beads in two separate tubes. In the procedure, the same sample was placed into each of 2 tubes.
• the 5 micron anti-kappa beads could be red and the 5 micron anti-lambda beads could be blue.
• the procedure could still be in 2 tubes as described above, or the kappa and lambda beads could be added simultaneously to the first tube. Analysis of this latter result would show a complex like Fig. 3 with blue only around the periphery (indicating monoclonal lambda), red only around the periphery (indicating monoclonal kappa), or a combination of red and blue around the periphery (indicating polyclonal B cells).
• Example 3 A 19 year old man presented with headache and stiff neck to the emergency.
• a major advantage of the invention is that analysis of cell populations can now be performed by simple inspection of the glass slide by any physician or technologist. This land of analysis can be used on any type of cell population bea ⁇ ng specific cell surface markers and in a wide va ⁇ ety of conditions (lymphoma is one example).
• Malignant clones from patients with acute leukemia can be similarly analyzed (using different types of markers), as can cell populations from patients with acquired immune deficiency syndrome.
• tumor markers for solid neoplasms become available, this kind of analysis can also be performed in a similar fashion.
• the new MN/CA9 antibody appears to be specifically expressed by dysplastic and malignant ute ⁇ ne cervical squamous cells. Since these cells may be suspended in a sea of normal cells, they may be difficult to identify even by routine lmmunohistochemistry. This method of analysis may both identify these cells and en ⁇ ch a cytological preparation for them so that they can be more easily analyzed.
• the present invention also provides a kit for practicing the invention.
• the kit contains one or more sets of beads as desc ⁇ bed above. Each set of beads is preferably in a container such as a sealed test tube. In some cases of simultaneous single parameter or multiparameter analysis, two or more sets of beads can be premixed, but typically they are kept separated.
• the kit also preferably contains one or more approp ⁇ ate filters as desc ⁇ bed above and preferably a set of instructions
• the methodology desc ⁇ bed herein can be automated and condensed. An example of a semiautomated device 25 for the performance of this kind of analysis is depicted in Figure 4.
• a sample is prepared to make a cell suspension
• the sample is then loaded into the machine 25 in the sample loader 26 and the machine 25 is programmed for the kind of analysis desired (lymphoma screen, acute leukemia analysis, myelodysplasia, etc ).
• the sample is divided into the approp ⁇ ate number of reaction chambers 28 (for example, 2, 4, 6, 8, 10 or 12) and a preprogrammed number of bead sets (for example, 1, 2, 3, 4, etc. bead sets) added sequentially or simultaneously to each reaction chamber.
• FIG. 5 is a schematic for a suggested lymphoma panel slide using such a procedure.
• Fig. 5 shows 6 wells, each having run a 3-bead set as shown for multiparameter analysis.
• CD20/kappa/lambda This indicates a well where the machine ran the CD20/kappa/lambda analysis described earlier herein.
• the other 5 wells give antibody information for running similar analyses as known in the art.
• a fourth or fifth set of beads can be added for further levels of analysis.
• the resulting complexes (such as in Fig. 3) are stained by immunohistochemistry or in-situ hybridization and then evaluated.
• Coated glass slides are preferred, to increase adhesiveness.
• the slides are stained, coverslipped and examined by routine light microscopy to assess binding.
• Cells bound to beads are preferably assessed to characterize and ensure cell type.
• cells in suspension in fixative or tissue media can be phenotyped by antibody coated beads and isolated from the surrounding milieu by the use of a filter of proper pore size. These bound cells, thus separated from the sea of other cells, can be transferred to a glass slide and stained with a variety of stains for visualization.
• a routine cytologic preparation using a variety of methods such as cytospin, cell block, or ThinPrep can be prepared.
• Single parameter analysis can be used to phenotype cells of interest, such as enumerating relative numbers of kappa and lambda-bearing B lymphocytes.

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